Enhanced oral bioavailability and bioefficacy of phloretin using mixed polymeric modified self‐nanoemulsions

Abstract Phloretin (Ph) is a natural active ingredient with wide biological properties. However, its poor water‐solubility and low oral bioavailability limit the application significantly in functional food and drug. This study was to explore the mixed polymer Pluronic® F127 and P123 modified the different triglycerides (LCT, MCT, SCT) in self‐nanoemulsions (SNEs) for enhancing the oral bioavailability and bioefficacy of Ph. The SNEs were characterized in terms of physical property study, lipolysis study, pharmacokinetic study, and anti‐inflammatory effect. The water‐solubility of LCT‐Ph‐SNE increased 3000‐fold compared with Ph solution. Pharmacokinetic study of SNEs and other carriers (HP‐β‐CD, PVP) results indicated that LCT‐Ph‐SNE was 7.9‐fold more bioavailable compared with unformulated Ph. The anti‐inflammatory activity of LCT‐Ph‐SNE in vivo represented a 6.8‐fold enhancement compared with unformulated Ph. This novel SNE formulation may also be used for other poorly soluble ingredients with high loading capacity, which made a significant impact on functional food and drug.

improve the solubility of phloretin effectively (Vo, Park, & Lee, 2013;Wei, Zhang, Memon, & Liang, 2017). And in recent years, the self-nanoemulsions (SNEs) have attracted increasing interest in food and drug research, due to its excellent ability to improve the solubility of hydrophobic drugs. It was widely used as a significant source of energy, essential fatty acids (FAs), and fat-soluble vitamins, which is a beneficial clinical effect for the terminally ill, pediatric, and longterm parenteral nutrition patients. Though the nanoemulsions were regarded to be a considerable carrier for the poorly water-soluble drugs, nevertheless it still has some potentially limiting factors that must be overcome. It was likely to be degraded in gastrointestinal (GIT), leading to prompt release and precipitation of encapsulated drugs, which removed by intestinal mucus, decreasing the delivery capability of SNEs. So the traditional SNEs need the further improvement, enabling them to be resistant to luminal digestion and capable of traversing the mucus layer rapidly. People have studied its stability about the oil phase, but few people were aware that the emulsifiers would significantly influence the drug's absorption, distribution, metabolism, and excretion in the body. Therefore, we need to search for an efficient self-nanoemulsion system for the hydrophobic compound (phloretin) to improve its bioavailability and bioefficacy.
In previous papers, it reported that the nanoemulsion with the F127 coating developed to minimize the degradation clearance by enzymes in mucus (Song et al., 2018;Yu et al., 2018). In this study, through the effect of the characteristics on in vitro and in vivo stabilities, we found that the effectiveness of SNEs with F127/P123 mixture coating at inhibiting drug digestion was strongly correlated to the physicochemical factors of the nanoemulsions. The physicochemical factors are including carbon chain length, steric hindrance, surface tension, concentration, charge, structure, lipid digestion, and activity of emulsions. Therefore, for more effective drug treatment, we not only study the function of the physical and chemical properties of Nanoemulsion, but also the function of how the rats body's response to the administration of water-soluble drugs. By measuring the changes in physical and chemical properties of different SNEs, we could have a better understanding of its interfacial behavior at oil-water interfaces. It may be altered after nanoemulsion formation using some approaches. The Ph solution and suspension (PVP-Ph) was chosen as the reference formulation. An in vitro digestion model was used to investigate the effect of lipid composition and type of formulation on drug solubilization and lipid digestion. The bioavailability of the drug from the tested formulations was investigated in rats. Finally, the comparison of anti-inflammatory effect to further prove that developed SNE formulations could improve drug solubility and oral bioavailability.
The purpose of this study was to develop SNE formulations intended to enhance the solubility and oral bioavailability of Ph and to compare the performance of different formulations in pharmacokinetic study. The oils were rapidly surrounded by surfactants, which were adsorbed to droplet surface lead to decreasing the interfacial tension; thus, we could found that the F127/ P123 polymeric mixture had highly surface active (Figure 1). The limited permeability of particles through mucus can further lead to their clearance from the GIT, resulting in poor absorption . It has been reported that nanoparticles (NPs) modified with polymers, which has a long hydrophilic chain, such as Pluronic® F127, P123 or polyethylene glycol (PEG) polymers, could exhibit excellent mucus diffusion ability. Therefore, we hypothesize that SNEs will efficiently overcome both lipases and mucus barriers if they are decorated with hydrophilic polymers.
In this study, the optimized SNE with improved physicochemical characteristics provides a more potent formulation of Ph as a therapeutic and functional food ingredient. Moreover, this work has important implications for the pharmaceutical food by using the optimized nanoemulsion systems.

| Preparation of the Ph-loaded selfnanoemulsions (Ph-SNEs)
Compositions of the Ph-SNEs are given in Table 1. Each SNE consists of ethanol, 1,2-propanediol, oil phase, and surfactant, (1:1: 4:6, w/w). Briefly, Pluronic P123 (25 mg) and Pluronic F127 (25 mg) was firstly dissolved in the mixture of co-solvents (50 mg ethanol and 50 mg 1,2-propanediol). Ph (400 mg) was dissolved in the mixture of oil phase (200 mg, SCT, MCT or LCT) and surfactant (200 mg, Cremophor EL) under stirring for 20 min at 50°C. The hot oil phase was dispersed in the water phase (the co-solvents of the ethanol, 1,2-propanediol) at the same temperature. These Ph-SNEs were prepared using emulsification followed by vibrating for 20 min at 50°C to make it homogeneity. The final concentration of Ph in the nanoemulsion was 275 mg/g. F I G U R E 1 A schematic diagram for (a) the design of SNEs and its bioefficacy study, (b) the transport process of the normal SNEs and Mixed-SNEs (F127/ P123 mixed polymer modified selfnanoemulsions) through the mucus layer and epithelial barrier The prepared nanoemulsions were diluted to 200 times with deionized water and mixed well. The particle size, PDI, and zeta potential were measured by the dynamic light scattering method (Nano ZS, Malvern Instruments Ltd, UK). The light source was at a fixed scattering angle of 90° at 25°C, with 633 nm and 30 mW power (Fan et al., 2011;Huang, Wu, Tu, Lai, & Liou, 2015). Meantime, to the evaluation of the storage stability, the samples were analyzed by mean droplet sizes with Zetasizer Nano after storage during storage (30 days) without light at room temperature (Yuting, Yuzhu, Wally, & Jiang, 2017).

| TEM morphology analysis
The morphology was observed by transmission electron microscopic (TEM) (HT7700, Hitachi, Tokyo, Japan). Then, it was diluted with 100 ml of deionized water and mixed well. The 10 μl of diluted nanoemulsions was stained with 1% phosphotungstic acid (PTA) for 1 min and then dropped on a copper grid for 2-3 min. The specimens were transferred to the TEM and analyzed at 100 kV after drying in air at room temperature.

| Solubility study
To determine solubility, an excess amount of sample was added into 10 ml distilled water. The samples were placed in a shaker and shaken for 24 hr at room temperature to attain equilibrium mixture.

| Optical determination of interfacial tension
The interfacial tensions were measured at the air-water interface of different nanoemulsions, using the pendant drop technique by a fully automatic contact angle analyzer (OCA100, Dataphysics, Germany).
An inverted nanoemulsion drop was formed at the tip of the needle fitted to a syringe with a total volume of 100 μl. The continuous droplets pushed out from the tip of needle between 30 and 10 μl. The video images of drop shape were captured by camera until the nanoemulsion drop detached from the tip of the needle due to the decreased interfacial tension. The drop shape in each image was analyzed by the contour analysis system (SCA22 software), based on the Young-Laplace evaluation of pendant drops (Donsi, Senatore, Huang, & Ferrari, 2010;Shu et al., 2018;Tran, Guo, Song, Bruno, & Lu, 2014).

| Apparent viscosity
Viscosity measurements (Pa·s) were performed by using a Vibro Viscometer (SV-10, A&D Company, Tokyo, Japan) vibrating at 30 Hz, with constant amplitude and working at room temperature. Aliquots of 10 ml of each nanoemulsion were used for determinations. During shear rate from 0 to 100 s −1 , apparent viscosity (Pa·s) was recorded (Lu, Xiao, & Huang, 2018).

| In vitro stability study
The in vitro stability study was measured by monitoring the particle size and PDI of SNEs in PBS (pH 7.4) and biorelevant media at 37°C for 24 hr using a water-bath shaker. The biorelevant media included simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). All the media were prepared as previously reported (Huang et al., 2015; Wilde, Garcia-Llatas, Lagarda, Haslam, & Grundy, 2019). The SGF was composed of 0.2% sodium chloride (NaCl) (w/v) and 0.32% pepsin (w/v); then, the pH was adjusted to 1.2 by 0.1 M HCl. The SIF was composed of 0.68% monobasic potassium phosphate (KH 2 PO 4 ) (w/v) and 1% pancreatin (w/v); then, the pH was adjusted to 6.8 by 0.1 M NaOH solution.

| In vitro lipolysis study
The lipolysis study was performed by an in vitro lipid digestion model as previously describe (Wilde et al., 2019). Briefly, 100 mg SNE was dispersed in 19 ml 50 mM Tri-maleate digestion buffer (pH 7.5). Digestion study started by adding 1 ml 10% (w/v) porcine pancreatic lipase solution (4,000 tributyrin units/ml). To neutralize the

| Pharmacokinetic study in rats
The pharmacokinetic study was investigated in Sprague Dawley rats (300-320 g) purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China) and the approval document is SCXK/2018-0002. The rates were kept for 7 days in the environmentally controlled room (12/12 hr dark/light cycle, 25 ± 2°C, 50% humidity) before the experiments study for acclimatization to animal house conditions. All rats were given free access to diet and water. All animal experimental protocol used in this study was ap- Before analyzing, plasma was thawed at 37°C. 0.2 ml sample was added to 0.2 ml acetonitrile and vortexed for 1 min. The mixture was centrifuged at 12,000 rpm for 5 min, and the upper phase was collected. Then, 20 μl sample was injected into HPLC for analysis on Ph concentration of plasma.
The column temperature was maintained at 30°C, and the mobile phase was 30% acetonitrile in water with a delivery flow rate of 1 ml/min. PDA detector (detection wavelength 286 nm) was used.
The pharmacokinetic parameters were calculated by the pharmacokinetic software WinNonlin Standard Edition v1.1 (Pharsight Corp., Mountain View, CA, USA). The Ph, Blank plasma + Ph, and plasma sample at 0.083h in rats were analyzed with HPLC based on the above chromatographic conditions. As shown in the Figure S1 is less than 2%.The mean extraction recovery was 82.02 ± 1.2%, 84.67 ± 1.5% and 81.51 ± 0.85% at 0.5, 10.0, 20.0 μg·ml-1, respectively. The limit of quantization (S/N = 10) was estimated to be 0.3 μg/ml, and the limit of detection (S/N = 3) of phloretin was estimated to be 0.1 μg/ml in plasma. It indicated good accuracy and precision of the developed method.

| Anti-inflammatory effect
The anti-inflammatory effect was carried out on the

| Statistical analysis
All data were presented as mean ± standard deviation (STD). Data were compared by independent Student's t test was used to compare the means of two groups. The level of significance was set at p < .05 and p < .01, which was considered as significant and highly significant. All the statistical analysis was done by using SPSS software (SPSS, Inc., Chicago, IL, USA, version 17.0).

| Droplet size, size distribution, and ζ-potential
The preparation of SNEs was based on a self-emulsifying system, and Ph was solubilized in the oily core of the nanoemulsion structures with F127& P123 mixture coating ( Figure 2  In previous work that the size and zeta potentials of SNEs without modification were around −20.0 mV (Xia et al., 2017). In this study, the zeta potentials of SNEs were in the range of −3.4 to −2.10 mV (close to neutral). The increase of zeta potential showed that the extension of the hydrophilic polyethylene oxide (PEO) chains of P123/ F127 mixture coated the oil phase surface, which had further increased the hydrophilicity of SNEs and shielding of droplets' surface charges. Moreover, PEG-modified nanoparticles with neutral surface charges undergo more rapid transport than those anionic charges in mucus (Lai, Wang, & Hanes, 2009). Therefore, we estimated that this optimized SNEs with near-neutral charge would appear rapid transport and then highly absorption by oral administration.

| TEM morphology analysis
As shown in Figure 4, the morphology of SNEs observed by a transmission electron microscope (TEM) after 24 hr in distilled water. All nanoemulsions (Figure 4b-d) loaded Ph were less than 100 nm in size and the spherical nature in shape. That is almost consistent with that obtained in the droplet size analysis in Table 2 However, the shape of the SNE without mixed polymeric modified is similarly oval (Figures 4a and 5).

| Solubility study, interfacial tension, and apparent viscosity
The result of the solubility study of Ph in various SNEs has been shown in Figure 5. The solubility of Ph in SCT-Ph-SNE, MCT-Ph-SNE, and LCT-Ph-SNE was 50 ± 4.5, 55 ± 4.2, 60 ± 5.0 mg/ml, respectively.
The Figure 6 illustrated that there was a small increase in solubility of TA B L E 2 The size, size distribution, and ζ-potential of the Initial SNEs and SNEs after 30 days

F I G U R E 3 (a) Size distribution (nm)
and ( size and higher viscosity (Yesiltas et al., 2018). Due to the long chain in the oil phase, the SNEs with higher apparent viscosity improved physically stable.

| In vitro stable study
Before conducting in vivo studies, it was necessary to ensure the stability of SNE nanoparticles. In this study, we used all kinds of buffers and biorelevant medias such as PBS, SGF, and SIF to investigate the stability of SNEs. As shown in Figure 8a-d, there were no significant changes in particle size and PDI observed in PBS and SGF buffers in 24 hr. This suggested that the structure of SNEs particles did not undergo obviously damaged; thus, it could make more resistance to the acidic conditions in the intestinal fluid (SIF). Nevertheless, particle size and PDI of SCT-Ph-SNE had increased dramatically in SIF (Figure 8ef), which meant that the structure of particles had damaged and then the mixed F127/P123 polymeric membrane had been broken down.
The SCT-Ph-SNE digested much easier than other SNEs by lipase in the intestinal environment, which was likely because of the shortchain oil. Hereby, it showed that the digestion rate was relying on the length of the fatty acid chains, the longer the fatty acid chains, the more efficient in delaying the digestion rate. Finally, we found the LCT-Ph-SNE was the most stable in vitro stable study.
Based on the above results, the SNEs were selected as a study subject to evaluate its pharmacokinetic study and bioefficacy study.

| In vitro lipolysis study
To simulate in vivo lipid biodegradation, in vitro lipolysis study commonly used by the alkaline compensation method (Han et al., 2009) though it is just an accelerated process. As shown in this study The least drug precipitation from LCT-Ph-SNE illustrated that the LCT-Ph-SNE was highly effective in inhibiting lipid digestion.

| Pharmacokinetic study
The principal goal of oral drug delivery is to improve the bio-  Table 3.
The AUC 0-24 hr and C max of all SNE formulations were much higher than that of the Ph solution, HP-β-CD-Ph and PVP-Ph (Ph quickly metabolized of Ph results in less T 1/2 . Additionally, we found that the Ph aqueous suspension displayed low drug bioavailability due to the low aqueous solubility of Ph and then the Ph would be easy to precipitate from the suspension. Take together the in vitro data, we could observe that the oral bioavailability is closely interrelated to the stability and physical properties of SNEs such as solubility study, interfacial tension, apparent viscosity and so on. That is consistent with previous researches about the emulsion (Arshad & Andreas, 2018;Yuting et al., 2017).
Overall, we observed that the optimizing SNEs system was quite effective to enhance oral bioavailability, compared with other carriers bioavailability of the SNEs system is more likely to inhibit digestion by enzyme and promoted mucus penetration, which led to high oral bioavailability (Song et al., 2018). It could be used as a possible formulation for poorly water-soluble agents to improve its oral bioavailability.

| Anti-inflammatory effect
To further prove that the above-mentioned data were good estimations in bioefficacy of the SNEs system, we observed the anti-  (Adami et al., 2012;Oliveira et al., 2017).
Then, the anti-inflammatory effect was evaluated in a TPA-induced rat ear edema model. It has been proved that increased skin edema is the first hallmark of skin inflammation including the process of increased vascular permeability and proliferation of epidermal keratinocytes (Nickoloff, Ben-Neriah, & Pikarsky, 2005). It can be seen from Figure 11 that TPA topical treatment alone results in a rat ear erythema and edema, and then, the average weight of TPA- to the more oral bioavailable through the SNEs system in pharmacokinetic study. Therefore, we continue studying for these SNEs by the below histological analysis.
Histological appearance evaluation has always been the appropriate way to assess the disorders of inflammatory tissue (Liu, Li, Zheng, Zhang, & Du, 2015). Then, we observed the effect of the Ph solution and the SNEs on histological appearances of rat ear. The histological appearances of the ear sections showed in Figure 12a that the normal group treated with water displayed the normal appearance in the epidermal layer without any obvious lesion. In contrast, the TPA topical treatment caused an obvious inflammation response with clear evidence of ear edema and inflammatory cell infiltration ( Figure 12b). As the suppression rate above, all SNEs could significantly suppress the signs of inflammatory responses (Figure 12d-f).
Nevertheless, in comparison with the inhibitory effects of these SNE formulations, the LCT-Ph-SNE showed the most decreased inflammatory cell infiltration and its effect is better than that of SCT-Ph-SNE and MCT-Ph-SNE (Figure 12f). Taken together with data of inhibition on ear edema and proinflammatory mediators, these results specify the best anti-inflammatory of LCT-Ph-SNE compared with the unformulated Ph. Additionally, in all SNEs samples, the LCT-Ph-SNE has the best anti-inflammatory activity, which may be a potential treatment of inflammation-associated diseases such as rheumatoid arthritis. And it should deserve more investigation because it may be a beneficial food for further development in clinical application.

| CON CLUS ION
In this study, we reasonably designed and investigated multifunctional SNE formulations for the oral delivery of hydrophobic components. The LCT-Ph-SNE had nearly neutral surface charge and better stability in different biorelevant media. The presence of a mixed hydrophilic group (F127/P123) on the surface of the oil droplet generated a steric hindrance, which is more likely to reduce enzymatic degradation in the gastrointestinal tract. Furthermore, efficient oral absorption of Ph was achieved by the LCT-Ph-SNE, with 6.8-fold higher AUC0-24 hr values compared with those of Ph.
Overall, we demonstrated that our SNEs were able to effectively enhance the oral bioavailability and bioefficacy of hydrophobic drug phloretin by the SNEs, while the mucus-penetrating ability of this formula needs further investigations based on current results.
Also, this novel SNE formulation may be used for other poorly soluble drugs, which will be a promising platform for oral delivery.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.